Metabolite of chiral cycloxaprid in solvent and in the raw of Puer tea

Highlights • The present study was performed with enantioselective degradation, transformation and metabolite of CYC in different solvents under light and raw Puer tea processing.• This degradation pathway in acetone under and Puer tea processing was firstly reported.


Introduction
Neonicotinoids is a valuable synthetic insecticides piercing-sucking pests of for tea protection (Tomizawa & Casida, 2003). They are used increasingly and occupied 24% of insecticides in world market (Jeschke et al., 2011). In 2013, the European Commission prohibited the use of three neonicotinoide insecticides (Thiamethoxam, imidacloprid and thiamethoxam) that might affect the life cycle of bees, which is urgent to assess other neonicotinoid's food safety risk (Chen et al., 2017). Li et al. 2011 reported that a new neonicotinoid insecticide of cycloxaprid (CYC) has been synthesized and applied in China. It is different from traditional neonicotinoids, which act as agonists of native and recombinant nicotinic acetylcholine receptors (nAChRs) (Liu and Casida, 1993, Matsuda et al., 1998, Nishimura et al., 1994, Tomizawa and Casida, 2003. Cycloxaprid is chiral compounds, which contains a chiral oxabridged cis-configuration (C ring) leading to a pair of enantiomers, 1R,2S-cycloxaprid and 1S,2R-cycloxaprid.
In recent years, chiral pesticides is the main focus of great attention at stereoselective biological activities and environmental processes of isomer selectivity. Chiral pesticides are commonly used as racemic mixtures and their stereoisomers are often degraded stereoselectively in food. Previous studies shown that the stereomiosmer had been found in soil or food. Liu et al. 2022 found the enantioselectivity of cycloxaprid in raw or ripen Puer tea processing. Zhang et al. 2013 observed stereoselective uptake and translocation of cycloxaprid in edible vegetables from roots. However, Chen et al. 2017 demonstrated that there was non stereoselective preference for cycloxaprid enantiomer in aerobic soils, so the enantioselectivity was effected by different matrix and the result was different. The degradation of pesticide is found the metabolite which maybe result in high toxicity in environment or food. Liu et al. 2015 identified and tracked 11 and one unknown transformed product in flooded and anoxic soil. Chen et al. 2017 reported three mainly metabolites included cleavage of the oxabridged seven-member ring and C-N between chloropyridinyl methyl and imidazalidine ring, carboxylation of the alkene group, and hydroxylation of imidazolidine ring in aerobic soil. Hou et al. 2017 studied photodegradation of CYC and analyzed 25 photodegradation products were identified via UPLC-TOF-MS/MS in water.
Puer tea is the most famous tea in China. Because its special processing, the Puer tea is fermented by traditional technology in longdistance transport with consign for horse. Previous our studied that one mainly metabolite was reported in Puer tea processing (Liu et al., 2022) but, the relevant information of degradation pathway and metabolite of CYC was still incomplete (Fig. 1). Therefore, it is important to obtain the degradation productions and pathway of CYC. The present study was to use racemate CYC to characterize the degradation in raw Puer tea processing, analyzed its metabolites and proposed degradation pathways, which is also significant for the fate of other new pesticides in the food.

Reagent
A racemic cycloxaprid was provided by Beilinwei technology Ltd. (Beijing, China). Cycloxaprid powder 25 % was supplied by shanghai shengnong pesticide Co Ltd (Shanghai, China). The purified materials of C18, PSA, Carb were supplied by Dima technology co. Ld. (Beijing, China).
The stock solutions were produced by dissolving the racemic cycloxaprid in acetonitrile. All solutions were stored in a refrigerator at − 18 • C. HPLC-grade acetonitrile and methanol were provided by Tedia Company Inc. (OH, USA). The initial dose of cycloxaprid 0.15 g/L was used with 0.6 g of cycloxaprid power completely dissolved into 1 L water.

Methods
Incubation experiments were performed to investigate the enantioselective racemic cycloxaprid and the metabolism in raw Puer tea processing. Five grams of raw Puer tea are sprayed with 0.15 g/L the formulation product solution. A nontreated control was also included. The enantioselective degradation is express as enantiomer fraction (EF) and determined by LC-MS/MS. The metabolomics are analyzed by LC-HRMS.
To research dissipation during light or dark, three hundred grams of raw Puer tea are sprayed with 0.15 g/L the formulation product solution. One sample is placed with black plastic bag on dark room, the other sample is not protected under air temperature. The samples are collected at intervals time on 0 (2 hr), 1, 3, 5, and 14 day. The residues amount expressed as dry sample.

Samples preparation
Two gram of tea sample were exactly weighed, then 10 mL water, 10 mL acetonitrile were added. After the mixture was vortexed, and added 4 g NaCl. The tube with vortex mixer was shaken vigorously at 1 min. The mixture was centrifuged at 5000 rpm for 5 min. The upper layer solution was separated and added by 100 mg PSA, 100 mg C18, 50 mg carb and 300 mg anhydrous MgSO 4 . After shaking and centrifugation at 5000 rpm for 3 min, 0.5 mL of the upper layer was separated and filtered through 0.22 μm filter for UPLC-HFMS or UPLC-MSMS.
The instrument was tuned in the positive ESI mode (3.8 kV of spray voltage, 325 • C of capillary temperature, 350 • C of probe heater temperature and 60 V of SLens). The instrument was calibrated using

Quality control
Cycloxaprid at three concentration levels (1, 10, 100 µg/kg) with matrix solution is used for limit of detection limit and recovery test. The chromatogram of spiked sample and elution order of racemic cycloxaprid was determined by online optical rotation. The more separation resolution was obtained by the method than Zhang et al. 2013 reported. No cycloxaprid is residue in the blank sample. The mean recoveries are in the range of 76-108 % for racemate of cycloxaprid, respectively. The corresponding relative standard deviation was 4.6-9.2%. The linearity is the range of 1-100 µg/L with coefficient, R 2 > 0.999. The limit of detections (LODs) with three times signal-to-noise (S/N) ratio, and the limit of quantify (LOQ) with ten times S/N ratio is 0.5 μg/L, 0.5 μg/kg respectively.

Data analysis
The data was obtained by triplicate samples. The results showed as the means with standard errors (means ± STD) using statistical analysis software. The significant difference (p = 0.05) among treatments was determined by one-way analysis of variance.

The stability of optical pure compounds in different solvent
The stability of optical 1R, 2S-cycloxaprid and 1S, 2R-(-)-cycloxaprid was tested in three solvents. The results showed that 1R, 2S-cycloxaprid or 1S, 2R-(-)-cycloxaprid in acetonitrile and acetone was stable over 17 day. However, optical pure of 1R, 2S-cycloxaprid was not stable in methanol, which was quickly decreased in methanol. But the racemate CYC was stable, the result showed that the transformation of 1S, 2R-(-)-cycloxaprid was formed in Fig. 2. A similar result is observed with other optical pure compounds (Zhang et al., 2013).
The parent compound of racemate CYC in solvent was more unstable under light than under dark. Hou et al. 2017 reported the pathways of the photoreaction process of CYC was hydroxyl radicals reaction. The degradation of CYC in three solvents was distinctly decreased under light. The CYC in acetone was not detected over 9 day, but an amount of residues in methanol, acetonitrile were founded. This suggest that the degradation of cycloxaprid was catalyzed by light radicals and speed by acetone solvent.

Formation of metabolites in acetone under light
The metabolite research is major limited with the relatively small number of metabolites commercially available as pure standards and structure determination by NMR or X-ray crystallography, therefore the large number of metabolites with unknown chemical structures that remain to be identified and characterized by High resolution mass spectrum (Ivana et al. 2019). Novel computational tools can predict MS fragmentation patterns in databases (Cajka et al. 2017;Kind et al. 2018), which has been shown that fragmentation spectra can be simulated with quantum chemical and molecular dynamics methods (Wishart et al., 2018). Fig. 3 shows the chromatogram of main metabolites under light with acetone at 13 day after cycloxaprid spiking. There were two large peaks with the retention times (T R ) at 34.83(g), 15.78(f) min, and four main peaks were named as a, b, d, e. We were achieved metabolite information using a combination of different tools. The metabolite compounds   were identified based on accurate mass and retention time using the NIST17, HMDB 20, GNPS 21, GMD and the Lipid MAPS libraries, which obtained the molecular structure. Then for MSI annotations, we used mzCloud (online second mass spectrometry),ChemSpider (online first mass spectrometry) accurate mass search services for validating and putatively annotating the metabolite feature using mass spectrometry ( Table 1). The fragment pathways of CYC were three in Fig. 4, One possible pathway was via the cleavage of the oxabridged ring (C ring). The second possible route was oxidated the C ring. The third possible route was removed of chloropyridinyl (A ring). The pathway was similar with Hou et al. 2017 reported the pathway of photodegradation in water.
The peak at the retention time of 17.72 min with the molecule ion at m/z 323.09033 [M + H] + and chlorine isotopic ion at m/z 325.08752 [M + H + 2] + was CYC. The base peak of characteristic ion at m/z 126.01065 and chlorine isotopic ion at m/z 128.00773 corresponding to chloropyridinylmethyl, which could be found in other metabolite. The other major fragments at m/z 276. 09681, 248.09485, 151.08655, 123.09183 exhibited the loss of NO 2 , NO 2 + CO, NO 2 + ClC 5 H 3 NCH, and CO + ClC 5 H 3 NCH, respectively. The Fig. 5 showed MS/MS spectra of CYC and main metabolite.
Metabolite g was the major metabolite in total ion chromatogram. The product ions 270.10032 [M + H] + containing odd nitrogen and chlorine isotopic ion 37 Cl, corresponding to the formula of C 12 H 16 O 2 N 3 Cl. The product ions 212.05803, 197.04741 produced via the loss of OC 3 H 6 and OC 3 H 6 -NH, which was identified as 1-[(6-chloropyrid-3-yl) methyl]-2-hydroxyl-imidazoline-tetrahydropyran, which probably is a rearrange reaction from metabolism j.
Metabolite k, l, j was probably a leading compounds of g. The l of product ion 307.09518 and the k of 305.07922 was reduced oxygen from the NO 2 of CYC, and the three metabolites containing product ions m/z 126.01054 resulted from the cleavage of the C -N bond linking the A + B rings. The retention time of metabolite j and k was at 45.66, 38.27 min respectively, but metabolite l was at 16.66 min, which indicated that metabolite l was polar compound with alcohols and metabolitea j, k were low polar compound with oxo-bridge structure in Fig. 4. The

Proposed degradation pathway under light
Based on the identified degradation products, the possible photodegradation pathways in aqueous solution under light irradiation were proposed. The tentative photodegradation pathways of CYC were shown in Fig. 6. The degradation of CYC in acetone solution under light via three possible pathways.
One main degradation pathway was via the reduce reaction of NO 2 to NO, and rearrange reaction to tetrahydropyran, which was not previously reported (Fig. 6) Two degradation pathways were via the cleavage of the oxabridge seven member ring and the whole C ring, resulting in the formation of primary compound. These pathway was very similar to that reported in the photodegradation of CYC under environment. (Hou et al. 2017).

Formation of metabolites in raw Puer tea
To obtain the transformation, the conversion of cycloxaprid is quickly generated by sun light and the normal fermentation in air temperature. Fig. 7 shows the main metabolites under raw Puer tea processing at 13 day after cycloxaprid spiking and blank. There were three obvious peaks with the retention times (T R ) at 13.97 (x), 19.61(h), 39.09 (y) min, and the diversity peaks were similar with these peak on metabolites in acetone under light (metabolite of l, k, j, i).
Metabolite × was the major metabolite reported by our previous  results (Liu et al., 2022). The metabolite and standard were simultaneously analyzed by LC-HFMS, and their retention time, molecular ion peak, and fragmentation pattern were in agreement with the synthesized structure standard of 2 -chloro-5 -[[-2 -(nitromethylidenc) imidazodin-1 -yl] methyl] pyridine (CAS 10336-63-4). A similar result was also observed by Chen et al. 2017. The result indicated that most CYC might transform to the active intermediate x. Furthermore, Shao et al. 2010 showed that CYC may be a slow-release reservoir for (nitromethylene) imidazole (x), which was considered as the final active ingredient. The mass spectrum of h that appeared at 19.61 min in the chromatogram yielded mass peaks of m/z 212.05819 [M + H] + , which corresponded to the formula of C 9 H 10 ON 3 Cl. The Fig. 8 showed MS/MS spectra of main metabolite. The base peak was more different with m/z = 128.02614 than with characteristic ion m/z = 126.01055. The result suggested that the pyridine ring was added hydroxyl group and oxygen rearrange reaction. Metabolite

Proposed degradation pathway under Puer tea processing
One degradation pathway was via the cleavage of whole C ring, resulting in the formation of mainly degradation compound X, which had been reported as the final active ingredient (Chen et al. 2017;Hou et al, 2017,;Liu et al. 2022). Metabolism X also underwent an elimination of nitromethylene and methylation of the imidazolidine ring, which produced h and w.
The other degradation pathway was via the cleavage of oxabridge seven member ring and reducing NO 2 , resulting in the formation of metabolism l, which was similar to degradation in acetone under light. Metabolism l also underwent an elimination of nitromethylene and rearrange reaction, which produced y. This pathway in Fig. 9 was very different to photodegradation in water (Hou et al., 2017) or our previously reported results (Liu et al., 2022). Compared to the photolysis, the metabolite of CYC was more complex in Puer tea processing. This study can provide insights to the fate of pesticide in the Puer tea processing.

Conclusion
The enantioselective degradation, transformation and metabolite of CYC in different solvents under light and raw Puer tea processing was detected. Chiral cycloxaprid in acetonitrile and acetone was stable, however the transformation of 1S, 2R-(-)-cycloxaprid or 1R, 2S-(-)-cycloxaprid was founded in methanol. The fastest degradation of cycloxaprid occurred in acetone under light, the degradation pathway was via the reduce reaction of NO 2 to NO, and rearrange reaction to tetrahydropyran. The degradation pathway under raw Puer tea processing was via the cleavage of whole C ring, and via the cleavage of oxabridge seven member ring and reducing NO 2 , then it underwent an elimination of nitromethylene and rearrange reaction (Table 2).      (7); 121.07616 (6)